Oligonucleotides possessing 5'-phosphate groups are useful for many purposes. For example, these oligonucleotides are valuable tools for gene construction (Modrich et al., J Biol. Chem., 1973, 248, 7502-7511); cloning (Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.); mutagenesis (Fritz, DNA Cloning: A Practical Approach, IRL Press, Oxford, 1985, 1, 151-163); the ligation chain reaction (Barany, PCR Methods and Applications, 1991, I, 5-16); and many other biological applications. The above-noted documents each are entirely incorporated herein by reference. Often, such oligonucleotides are prepared by T4 kinase catalyzed phosphorylation employing adenosine 5'-triphosphate as a phosphate source (Sambrook et al., supra.).
A number of methods have been reported that allow chemical 5'-phosphorylation of pre-assembled oligonucleotide precursors. Some of them include preparation of modified nucleoside-based building blocks to be attached at the last step of the oligonucleotide synthesis (Bannwarth et al., Helv. Chim. Acta, 1990, 73, 1139-1147; and Tanaka et al., Nucleic Acid Chem., Wiley: N.Y., 1991, 4, 314-319; each of these documents is entirely incorporated herein by reference). Another strategy, based on non-nucleosidic building blocks, seems to be more universal, because a single reagent may be employed. A variety of approaches compatible with phosphotriester (Gorn et al., Bioorg. Khim. (Moscow), 1986, 12, 1054-1063), H-phosphonate (Gaffney et al., Tetrahedron Lett., 1988, 29, 2619-2622; and Marsters et al., Nucleosides Nucleotides, 1990, 9, 1079-1086), methyl phosphonamidite (Bhan, Tetrahedron Lett., 1944, 35, 4905-4898), or phosphoramidite (Horn et al., DNA, 1986, 5, 421-426; Wosnick et al., Gene, 1987, 60, 115-127; and Robertson et al., Nucleic Acids Res., 1989, 17, 9649-9660) chemistry have been elaborated. Each of the above-noted documents is entirely incorporated herein by reference.
All of these approaches suffer from the same shortcoming: the efficiency of the final coupling cannot be monitored by dimethoxytrityl response. In order to overcome this problem, a building block derived from (4,4'-dimethoxytrityloxyethyl) hydroxyethyl sulfone has been introduced (Horn et al., Tetrahedron Letters, 1986, 27, 4705-4708, which article is entirely incorporated herein by reference). See also U.S. Pat. No. 5,252,760, which patent is entirely incorporated herein by reference. Upon completion of the final chain elongation step, the 5'-derivatized oligonucleotide possesses a 5'-terminal tether containing a dimethoxytrityl protecting group. This group can be used to determine the coupling yield by conventional dimethoxytrityl assay. Ammonolytic deprotection, however, results in .beta.-elimination of the O-phosphorylated hydroxyethyl sulfone fragment, and hence the dimethoxytrityl group is lost on release of the 5'-phosphate group. Accordingly, performing dimethoxytrityl specific isolation of the oligonucleotide is excluded.
It is well known that preparative reverse phase ("RP") separation of oligonucleotide 5'-phosphates from the corresponding non-phosphorylated material is often unsuccessful. The use of more efficient ion exchange chromatography is, in turn, restricted by the length of the DNA fragment to be isolated. Therefore, approaches that offer a selective isolation of the desired oligonucleotide remain to be of particular interest.
A family of methods, all involving an "orthogonal" protection strategy of the 5'-terminal phosphate, has been elaborated. After the chain assembly and ammonolytic deblocking, the orthogonal protection of the 5'-phosphate t-butyl (Coe et al., Chem. Ind, 1989, 724-725; and Sekine et al., Tetrahedron Lett., 1991, 32, 395-398.), (4-nitrophenyl)ethyl (Uhlmann et al., Tetrahedron Lett., 1986, 27, 1023-1026; Uhlmann et al., J Chem. Scr., 1986, 26, 217-219; Schwarz et al., Nucleosides Nucleotides, 1987, 6, 537-539; and Bower et al., Nucleic Acids Res., 1987, 15, 3531-3547), 2-(tritylthio)ethyl (Connolly et al., Nucleic Acids Res., 1985, 13, 4485-4502; and Connolly, Tetrahedron Lett., 1987, 28, 463-466), or 2-(triphenylsilyl)ethyl (Celebusky et al., J Org. Chem., 1992, 57, 5535-5538)! remains unchanged. These documents also are each entirely incorporated herein by reference. Being of moderate hydrophobicity, the t-butyl and (4-nitrophenyl)ethyl protecting groups enable efficient separation only for relatively short oligonucleotides. In contrast, the 2-(tritylthio)ethyl or 2-(triphenylsilyl)ethyl groups are more hydrophobic than the dimethoxytrityl group, and hence the desired oligonucleotides may be very selectively isolated by reverse phase, high performance liquid chromatography ("RP HPLC") or reverse phase cartridge purification. After the purification, free 5'-phosphate monoester is released by treatment with an appropriate reagent, such as trifluoroacetic acid or trimethylsilyl chloride for t-Bu; strong amine (DBU, TBD, and others) for (4-nitrophenyl)ethyl; or 2 M Bu.sub.4 NF/DMSO at 70.degree. C. for 2-(triphenylsilyl)ethyl. In this respect, the 2-(tritylthio)ethyl group offers the mildest deprotection technique. To obtain the 5'-phosphate, the trityl-S bond was selectively cleaved by aqueous silver nitrate or iodine. Subsequent addition of dithiothreitol at pH 8.5 generates in both cases mercaptide ion which rapidly degrades to the target oligonucleotide 5' phosphate and ethylene sulfide.
There is a need in the art for reagents and an efficient process for producing high purity phosphorylated oligonucleotides. Preferably, this method would use reagents that are commonly available and used in DNA synthesis.